Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

A kit for detecting bovine circovirus and a RT-PCR detection method

A technology of RT-PCR and detection method, which is applied in the field of PCR detection, can solve the problem that there is no specific treatment for this disease, and achieve strong sensitivity, wide application prospects, and good repeatability

Inactive Publication Date: 2019-01-15
INST OF ANIMAL HUSBANDRY & VETERINARY MEDICINE HENAN ACAD OF AGRI SCI
View PDF0 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There is currently no effective treatment for this disease

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • A kit for detecting bovine circovirus and a RT-PCR detection method
  • A kit for detecting bovine circovirus and a RT-PCR detection method
  • A kit for detecting bovine circovirus and a RT-PCR detection method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Embodiment 1: bovine torivirus RT-PCR detection method

[0030] 1. Design and synthesis of primers for detection of bovine toroidal virus

[0031] Homology comparison analysis was carried out on the sequence of bovine torivirus published in GenBank, the sequence of Breda virus S gene with accession number NC_007447 was selected, and a pair of specific primers were designed by using Oligo 6.0 software. The nucleotide sequence of the primer is an upstream primer: 5'-GGTGCCGTTGTTGTGTCA-3', and a downstream primer: 5'-CAGCCCTGAGTTGCCTTATC-3'. The primers are expected to amplify a fragment size of 698bp.

[0032] 2. Clinical sample processing and virus nucleotide extraction

[0033] 133 feces collected from cattle farms in Henan area were used as clinical samples. Clinical samples were suspended in PBS at 1:10 (w:v), vortexed for 30s, then centrifuged at 8000r / min at 4°C for 5min, the supernatant was collected and stored at -80°C.

[0034] The total RNA was extracted usi...

Embodiment 2

[0044] Embodiment 2: specificity test

[0045] Utilize the RT-PCR method of detecting bovine torovirus that the embodiment of the present invention 1 constructs to bovine torovirus (BToV), bovine coronavirus (BCoV), bovine norovirus (BNoV), bovine astrovirus (BAstV), Bovine rotavirus (BRV), bovine viral diarrhea virus (BVDV), and bovine crest virus (BKV) are detected, and sterile double distilled water is set as a negative control simultaneously to verify the specificity of the RT-PCR method established by the present invention , the specific method is as follows:

[0046] Bovine torovirus (BToV), bovine coronavirus (BCoV), bovine norovirus (BNoV), bovine astrovirus (BAstV), bovine rotavirus (BRV), bovine viral diarrhea virus (BVDV), The RNA of bovine crest virus (BKV) was reverse transcribed to obtain the cDNA of each virus. Then use the cDNA of each virus as a template, carry out PCR amplification according to the optimized PCR amplification reaction system and reaction pr...

Embodiment 3

[0047] Embodiment 3: sensitivity test

[0048] Use the above primers to perform RT-PCR amplification on the positive samples of BToV, recover the PCR amplification products, and connect them to the pMD18-T vector respectively to construct positive plasmids, and use an ultra-micro nucleotide protein analyzer to determine the pMD 18-T-BToV positive The concentration of the plasmid is 50.5ng / μL, according to the formula (6.02×10 23 copy number / mole)×(nucleotide concentration ng / μL×10 -9 ) / (nucleotide length×660)=standard plasmid copy number copies / μL, the DNA copy number of bovine ringqu virus positive plasmid is calculated to be 1.36×10 10 copies / μL; the obtained positive plasmid was sequentially diluted 10 times (the dilution factor was 10 1 、10 2 、10 3 、10 4 、10 5 、10 6 、10 7 、10 8 、10 9 ), then respectively take the bovine ringqu virus positive plasmid DNA of each concentration gradient after dilution as a template, carry out PCR amplification according to the optim...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention discloses a kit for detecting bovine circovirus, which comprises a primer for detecting bovine circovirus, an RNA extracting reagent, a reverse transcription reagent, a positive controland a negative control, wherein the nucleotide sequence of the primer is an upstream primer P1: 5 '-GGTGCCGTTGTTGTGTCA-3 ', downstream prim P2: 5'-CAGCCCTGAGTTGCCTTATC-3'; Positive control is BCoV, BNoV, BAstV, BRV, BVDV and BKV, negative control is sterile double distilled water. The invention also discloses an RT-A PCR method comprises the following steps: (1) synthesizing the primers for detecting bovine circovirus; (2) extracting RNA from the sample to be tested, and reverse transcribing the extracted RNA as a template to obtain cDNA; (3) PCR amplification reaction is carried out by usingthe cDNA prepared in the step (2) as a template and the primer set synthesized in the step (1); (4) PCR amplification products are analyzed. Simple, practical, economical and efficient, it can greatlyimprove the detection efficiency and save the detection time.

Description

Technical field: [0001] The invention belongs to the technical field of PCR detection, and in particular relates to a RT-PCR detection method and application of bovine toroidal virus. Background technique: [0002] Bovine torovirus (BToV) is a member of the genus Torovirus in the family Coronaviridae of the order Nidovirales. The genome is a single-stranded positive-strand RNA with a size of about 25-30kb. The virus was first isolated in the feces of calves with diarrhea in the United States in 1979, and it was named Bredavirus (BToV). BToV has a history of nearly 40 years since it was first reported, and it is widely distributed in the world. The detection rate of BToV in the feces of calves with diarrhea has reached 2.9-36.4% worldwide, but there is no report about BToV in my country. , is still in a technical blind spot in the BToV field, and there are no effective measures for the prevention and treatment of bovine torovirus infection in cattle. [0003] After the catt...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
IPC IPC(8): C12Q1/70C12Q1/686C12N15/11
CPCC12Q1/686C12Q1/701C12Q2521/107C12Q2565/125C12Q2545/101
Inventor 师志海兰亚莉王璟王文佳焦文强徐照学孟红丽王亚州
Owner INST OF ANIMAL HUSBANDRY & VETERINARY MEDICINE HENAN ACAD OF AGRI SCI
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com